Ontogeny of the germline in non rodent mammals

A better knowledge of how the precursors of the gametes (the cells that make egg and sperm) develop in mammals has important implications for our understanding of animal development, with important applications in biotechnology, assisted reproduction and regenerative medicine.
This project will investigate the developmental program of the precursors of the gametes, the primordial germ cells (PGCs), in mammals. Knowledge of mammalian PGCs originates primarily from studies in mice, but very little is known about this process in other mammals. In mice PGCs are set aside very early in development in response to inducing signals from the extraembryonic ectoderm (ExE), a derivative of the trophectoderm. However, the mouse embryo is anatomically different to most other mammals at this stage of development. The mouse embryo (epiblast) undergoes cavitation forming an egg cylinder containing the ExE. In other mammals the ExE does not exist, and the epiblast forms a flat disc of cells. This raises the question of how and when the PGCs originate in humans and other non-rodents mammals lacking the structures (the ExE) known to play a critical role in PGC induction in rodents. We hypothesize that the differences in embryo development underlie differences in the mechanisms of PGC specification in mammals. We will test this hypothesis by investigating PGC development in pig embryos, as they share similar anatomical and developmental features to early human embryos, which are not available for research. Discoveries made in this species can therefore be translated to humans.
The aim of this project is to establish the genetic and epigenetic mechanisms of pig PGC development in vivo, followed by functional experiments with cultured isolated epiblasts to identify key master regulators of germ cell development. This knowledge will then be exploited to study human PGC development from human embryonic stem cells (hESC). These investigations will also allow us to determine the relationship between pluripotency and PGC development, which will underpin the development of new culture conditions for the establishment of pluripotent embryonic germ cells.
The objectives of the project are:
1- To establish the gene expression profile and epigenetic reprogramming of early pig PGCs. These experiments will define the genetic and epigenetic signature of these cells and will inform on strategies for functional evaluation of PGC determinants.
2- To identify key master regulators that promote PGC differentiation from pig epiblasts and from hESC and pEpiSC/piPSC. These studies will establish the functional role of PGC-master genes during pig (in vivo and in vitro) and human (in vitro) embryonic development, and inform on new strategies for the differentiation of PGC precursors from pluripotent cells.
3- To develop culture conditions for the establishment of pig embryonic germ (EG) cells, the in vitro pluripotent derivatives of PGCs. These experiments will use the knowledge gained from previous objectives to establish pluripotent and germline competent pig EG cells.
Insights into primordial germ cell formation will contribute to the development of methods for deriving new sources of pluripotent cells and for improving protocols of differentiation of gamete precursors and somatic cells in the laboratory. An important application of these technologies is in regenerative medicine and assisted reproduction. In addition, new sources of pluripotent cells in domestic animals are of great importance for increasing the efficiency of transgenesis.
This project addresses questions of strategic relevance to the BBSRC, such as lifelong health and well-being. We anticipate that the outcomes of the current project will contribute to the academic and clinical advance in the areas of regenerative medicine and global food security.

The germ cell lineage ensures the continuity of species by the creation of new progeny. In mice, primordial germ cells (PGCs) are specified from proximal epiblast cells in response to inductive signals from the adjacent extraembryonic ectoderm (ExE). Very little is known, however, about how PGCs develop in other mammals. Significant anatomical differences exist between rodent and non-rodent embryos at these developmental stages. Non-rodent embryos (including human, pig, etc.) do not have an ExE and the epiblast forms a flat disc instead of the egg cylinder typical of mice. Furthermore, these embryological differences suggest that the mechanisms of PGC specification could be under different regulation in mice compared to other non-rodent species. This project will investigate the ontogeny of primordial germ cells in the pig, as model for mammals with similar embryology.
To investigate this process we will perform the following experiments:
1- Establish the gene expression profile of early and late PGCs isolated from pig embryos.
2- Study the epigenetic reprogramming of pig PGCs at different stages of development.
3- Perform functional experiments in isolated pig epiblasts, hESC and pigEpiSC/piPSC to test the role of identified candidate genes or signalling pathways in PGC induction in vitro.
4- Establish the culture conditions required the establishment of pig EG germ cells.
These experiments will enable the identification of critical molecular determinants of pig PGC development in vivo, and provide the basis for the investigation of how PGC can be induced in vitro from human and pig pluripotent cells. Furthermore, because of the close relationship between pluripotency and PGCs, the new understanding of how PGCs form in the pig embryo will inform on new approaches for the generation of EG cells. In addition, because of the close similarities with the human embryo, this research will be important for understanding human PGC development.

Who will benefit from this research?
Advances in the field of gamete and stem cell biology will benefit the areas of health science, regenerative medicine, assisted reproduction, pharmaceutical industry and biotechnology. These areas are within the strategic priorities of the BBSRC in Lifelong Health and Wellbeing and Global Food Security.
The key impact areas of this research are:
1. Advance in methodologies for in vitro production of gametes which are required by the pharmaceutical and medical industries as tools for drug screening and toxicological studies. These in vitro produced gametes will be useful for 1- screening for effects of environmental pollutants in folliculogenesis, 2- screening for compounds that can modulate gamete development and for the treatment of infertility, 3- as a source oocytes with the capacity to reprogram somatic cells to pluripotency, with important applications in regenerative medicine. These developments will be of interest to regulators that will need to legislate for the use of in vitro gametes in humans. The pharmaceutical industry will be a primary beneficiary of this new developments. The applicants have established links with industrial partners (EvoCell, CellCentric, and Zoetis) and will seek to develop these technologies further in partnership with them.
2. Clinicians working in the area of cancer, in particular those related to paediatric cancers will benefit from our research that will contribute to advancing the understanding of germ cell tumourigenesis. This new understanding will lead to a better diagnosis and treatment.
3. Progress in the derivation of embryonic germ cells (EG) in farm animals is also of great biotechnological importance for increasing the efficiency of genetic modification of animals. EG cells represent a novel source of pluripotent cells available for transgenesis. This development will be of great benefit to scientists requiring animal models of human diseases such as Parkinson's disease, cystic fibrosis, and multiple sclerosis.
4. The biotech and pharmaceutical industries will benefit from having effective methods for generating transgenic large animals using pluripotent cells. There is strong demand for precise genetic engineering of animal cells for evaluation of the role of specific genes in muscle hypertrophy and fat deposition in pigs. The applicant has a collaborative project with Zoetis to develop such technologies, and the outputs of the current project will serve as a basis for future research funding in this area. We anticipate that these discussion will take place during the second half of the project.
How will they benefit from this research?
The application of our research could impact reproductive health, by increasing reproductive potential of infertility patients, and paediatric germ cell tumours diagnosis and prognosis. Our research will establish the principles of gamete development in the growing embryo. The insight into these developmental mechanisms will contribute to 1- establish novel approaches for in vitro gamete production and their use in assisted reproduction, and 2- facilitate the development of novel diagnostic and treatment approaches for paediatric germ cell tumours. Conversion of gamete precursors into pluripotent cells in vitro will also facilitate animal transgenesis, a much needed tool for the generation of genetically modified animals.
What will be done to ensure that they benefit from this research?
The applicant has agreements in place with the University of Nottingham spin-off company EvoCell Ltd. to commercialize the research. During the course of the project regular meetings (twice a year) between the Business Development Executive of the School of Biosciences, patent lawyers from the University (Nottingham and Cambridge) and representatives from EvoCell will be organised to discuss patentable/commercial possibilities.